A few years ago, three novel murine homeobox genes (Six1, Six2, and Six3) closely related to the Drosophila sine oculis (so) gene were isolated; all are included in the Six/so gene family. Because of its early expression in the developing eye field, Six3 was initially thought to be the functional orthologue of the Drosophila so gene. This hypothesis was further supported by the demonstration that ectopic Six3 expression in medaka fish (Oryzias latipes) promotes the formation of ectopic lens and retina tissue, and by the finding that in humans, mutations in Six3 lead to holoprosencephaly. This grant application proposes a multidisciplinary approach to elucidate the functional and molecular events controlled by Six3, which will serve as an entry point to unravel some of the early steps leading to visual system development. A combination of functional genetics, molecular biology, and developmental biology studies has been designed to address important hypotheses in three specific aims. Morphologic and detailed molecular analyses will be performed to precisely characterize the phenotypes resulting from the targeted disruption of Six3 in mice, which was accomplished toward the end of the last granting period (Specific Aim 1). In addition, a conditional knock-out approach will be used to determine the tissue-specific roles of Six3 in each of the structures (lens, retina, and ventral forebrain) that normally express this gene (Specific Aim 1). The functional role, and the in vivo significance of the protein interactions that have been uncovered for Six3 during the last granting period will be characterized, and additional partners that interact with Six3 in vivo will be identified (Specific Aim 2). Finally, transgenic mice, in ovo electroporation, and results generated from microarray analysis will be used to identify other components of the Six3 regulatory pathway and to study the functional roles of some of these proteins in this process (Specific Aim 3). The proposed studies will generate new information concerning the mechanisms of Six3 function during early murine development and will generate several useful animal models for the study of different aspects of visual system development and some pathological conditions.